Many epidemiologic studies have reported that dietary flavonoids provide protection against cardiovascular disease. Quercetin, a member of the bioflavonoids family, has been proposed to have anti-inflammatory, anti-atherogenic, and anti-hypertensive properties leading to the beneficial effects against cardiovascular diseases. Recent studies demonstrated that orally administered quercetin appeared in plasma as glucuronide-conjugated forms in rats and humans. Therefore, we examined the effect of chemically synthesized quercetin glucuronide on platelet-derived growth factor (PDGF)-induced cell migration and kinase activation in cultured rat aortic smooth muscle cells (RASMCs). PDGF-induced RASMC migration was inhibited by quercetin 3-O-beta-D-glucuronide (Q3GA). Q3GA also attenuated PDGF-induced cell proliferation in RASMCs. PDGF activated extracellular-signal regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, and Akt in RASMCs. PDGF-induced JNK and Akt activations were suppressed by Q3GA, whereas ERK1/2 and p38 MAP kinase activations were not affected. We also confirmed that PDGF-induced JNK and Akt activations were inhibited by antioxidants, N-acetylcysteine and diphenyleneiodonium chloride, in RASMCs. These findings suggest Q3GA would be an active metabolite of quercetin in plasma and may possess preventing effects for cardiovascular diseases relevant to vascular smooth muscle cell disorders.

Osteoblasts produce prostaglandins in response to a wide variety of stimuli. Induced prostaglandin synthesis is generally the consequence of elevated cyclooxygenase-2 (COX-2) expression. Agents as diverse as serum, bFGF, PDGF, PGE(2), or [TNFalpha + IL1beta] rapidly induce expression of COX-2 protein in murine MCc cells. Transient transfection studies using reporter constructs containing either wild-type COX-2 regulatory sequences or mutated cis-acting sequences linked to a luciferase reporter gene identify a CRE site and two NF-IL6 (C/EBP) sites which play important roles in the regulation of COX-2 expression in response to all these agents in osteoblasts. Induction of wild-type COX-2 reporter gene expression in MCcells by all these agents involves signaling through the MEKK/JNK pathway and activation of both c-Jun and the C/EBP family of transcription factors.

OBJECTIVE

After endothelial injury, smooth muscle cells (SMCs) in the arterial media are modified from a contractile to a sympathetic phenotype. This process includes a prominent structural reorganization and makes the cells able to migrate into the intima, divide, and secrete extracellular matrix components. A similar change occurs in culture and then in vitro system has been established as a useful model in which to study the control of SMC differentiation. The purpose of this study was to analyze the expression of a number of phenotype- and proliferation-related genes in vascular SMCs during the first week in primary culture.

METHODS

SMCs were enzymatically isolated from rat aorta and seeded on substrates of fibronectin (an adhesive plasma protein) and laminin-collagen type IV (two major basement membrane proteins) in a serum-free medium or in uncoated dishes in a serum-containing medium. Total RNA was isolated from the cells after different times of culture and analyzed by Northern blotting for expression of specific gene transcripts. In part, expression of the corresponding proteins was also explored by Western blotting and indirect immunofluorescence microscopy.

RESULTS

The results indicate that the proto-oncogenes c-fos, c-jun and c-ets-1 were already activated during the isolation of the cells and then continued to be strongly expressed for a few days. Especially in the serum-free groups, there was also early activation of the genes for the matrix metalloproteinases, stromelysin (MMP-3) and type IV collagenase (MMP-2). In parallel, an increased expression of the genes for two extracellular matrix components was observed, with an early rise in osteopontin mRNA and a later rise in collagen type I mRNA. At the end of the test period, the corresponding proteins were deposited around the cells in a fibrillar pattern. Among the matrix receptors investigated, the beta 1 integrin subunit showed a high and persistent expression, whereas the alpha 5 and alpha 1 integrin subunits showed lower and more variable mRNA level. In support of the existence of an autocrine or paracrine platelet-derived growth factor (PDGF) loop, an early rise in expression of the PDGF A-chain gene and a subsequent rise in expression of the PDGF alpha-receptor gene were noted.

CONCLUSIONS

It is proposed that the coordinated shift in gene expression here described to take place in connection with the phenotypic modulation of vascular SMCs in primary culture is part of a predetermined genetic program that normally serves the function to engage the cells in a wound healing response.

The expression of the EGF receptor, c-erbB-2 and PDGF receptor proteins has been studied in a series of human brain tumour biopsies and cell lines. Western blotting was used to determine the amount of protein present and their intrinsic and ligand promoted enzyme activities were studied by immunoprecipitation followed by autophosphorylation. EGF receptors were found to be expressed at very high levels in 40% of primary tumour biopsies, but at uniformly low levels in tumour derived cell lines. The c-erbB-2 protein was not detected in tumour biopsies, but was present at variable, but low levels in extracts of tumour cell lines. PDGF receptors were also found at moderate to low levels in both primary tumours and cell lines. The EGF receptor gene was amplified in four out of 14 primary tumours and this generally correlated with high levels of protein expression. The c-erbB-2 gene was not amplified. Employing the polymerase chain reaction and sequence specific oligonucleotides as probes there was no evidence of mutations in the c-erbB-2 gene transmembrane region. These results suggest that alterations of expression of the EGF receptor may play a role in human brain tumours. There was however no evidence for aberrant expression of the c-erbB-2 protein. Additional experiments are required to assess the influence of PDGF receptor expression in brain tumour cells.

We developed a novel method to quantify trace amounts of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) using antibodies against PIP and PIP2. With this method, polyphosphoinositides can be measured in the range from 20 to 500 pmol. We applied the method to quantify changes in PIP and PIP2 levels in Balb/c/3T3 cells stimulated by platelet-derived growth factor (PDGF) and prostaglandin F2 alpha (PGF2 alpha), growth factors that stimulate the hydrolysis of PIP and PIP2. PIP2 content decreased rapidly to about 60% of control within 1 min while PIP content decreased gradually but significantly to 60% (PDGF) or 70% (PGF2 alpha) of control. Simultaneously we measured the mass levels of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol (DG). Inositol 1,4,5-trisphosphate levels rapidly increased and reached a maximum at 30 s after PDGF or PGF2 alpha stimulation and then decreased to the control level within 2 min. On the other hand, DG formation showed biphasic changes. In the first phase, DG rapidly accumulated and reached a maximum at 30 s after PDGF or PGF2 alpha stimulation and then quickly decreased. In the second phase, DG accumulated gradually, but very markedly, 2 min after PDGF or PGF2 alpha stimulation. Considering the changes in PIP2, DG in the first phase seems to be derived mainly from PIP2 while most of the DG in the second phase derived from other lipids.

Treatment of cultured rat oligodendroglial progenitors with either platelet-derived growth factor (PDGF) or fibroblast growth factor-2 (FGF-2) activated extracellular signal regulated kinase 2 (ERK2). Activation was transient in response to PDGF, whereas it was greater and more prolonged in response to FGF-2. ERK2 activation by PDGF was preceded by a very rapid, robust and transient tyrosine phosphorylation of the PDGF receptor. Although there was consistently more activation of ERK2 in response to FGF-2 than to PDGF, immunostaining of FGF receptors 1 (FGFR1) and 2 (FGFR2) and their tyrosine phosphorylation in progenitors was very weak, and both receptors were up-regulated during differentiation to oligodendrocytes. Tyrosine phosphorylation of the FGF receptors was maximal from 15 to 60 min of treatment and was sustained for many hours. Binding of radioiodinated FGF-2 to FGFR1 was predominant in progenitors, whereas binding to FGFR2 was predominant in oligodendrocytes. ERK2 activation by PDGF was more sensitive to inhibition of tyrosine kinases, whereas ERK2 activation by FGF-2 was relatively more sensitive to inhibitors of protein kinase C. These differences in signal transduction pathways probably contribute to the different cellular responses of oligodendroglial lineage cells to PDGF and FGF-2, respectively.

The differing signal transductions elicited by three isoforms of platelet-derived growth factor (PDGF) were studied in cultured rat vascular smooth muscle cells (VSMC), which show different mitogenic responses to the three PDGF isoforms. PDGF-BB elicited a variety of cellular signals, including the phosphorylation on tyrosine of phospholipase C-gamma 1 (PLC-gamma 1) and the PDGF receptor, formation of inositol 1,4,5-trisphosphate and diacylglycerol, degradation of phosphoinositides (phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate) and elevation of intracellular calcium ([Ca2+]i). However, PDGF-AB failed to show some of these signals, although it stimulated [3H]thymidine incorporation to almost the same extent as PDGF-BB. Importantly, phosphorylation on tyrosine of the PLC-gamma 1 was far less (< 6.25%) in the case of PDGF-AB than that of PDGF-BB when assessed by immunoblotting. On the other hand, calcium ion entry from the extracellular medium was comparable in PDGF-AB- and PDGF-BB- stimulated VSMC. PDGF-AA, which did not stimulate [3H]thymidine incorporation, failed to show any of these effects with the exception of diacylglycerol formation. These observations suggest that the three PDGF isoforms stimulate different signal transduction pathways and that calcium ion entry, but not tyrosine phosphorylation of PLC-gamma 1, is essential for PDGF-induced mitogenesis in VSMC.

The neuronal glutamate transporter, EAA<em>C</em>1 (excitatory amino acid carrier 1), undergoes rapid regulation after treatment with platelet-derived growth factor (<em>PDGF</em>) or phorbol ester in <em>C</em>6 glioma cells and neurons. A large intracellular pool of EAA<em>C</em>1 exists, from which transporters are redistributed to the cell surface in response to these signals. Here we show that <em>PDGF</em> had no effect on subcellular localization of the glial glutamate transporter, GLT-1, after transfection into <em>C</em>6 glioma cells. <em>C</em>himeras consisting of domains from EAA<em>C</em>1 or GLT-1 were used to investigate structural motifs involved in <em>PDGF</em>-dependent redistribution of EAA<em>C</em>1. <em>PDGF</em> did not induce trafficking of an EAA<em>C</em>1 chimera containing the carboxyl-terminal domain of GLT-1; however, it did induce trafficking of a GLT-1 chimera containing the carboxyl-terminal domain of EAA<em>C</em>1. A truncated mutant of EAA<em>C</em>1 lacking 10 carboxyl-terminal amino acids was responsive to <em>PDGF</em>, whereas a mutant lacking 20 residues was not. Alanine substitution mutagenesis in this region revealed a short motif, (502)YVN(504), necessary for regulated trafficking. This motif was also involved in protein kinase <em>C</em>-dependent trafficking, as mutant transporters exhibited an attenuated response to phorbol ester. Interestingly, the presence of YVN in the homologous region of a nonresponsive chimera was not sufficient to confer regulated trafficking; however, the presence of a 12-amino acid motif starting at this Tyr residue was sufficient to confer responsiveness to <em>PDGF</em>. These studies identify a novel motif within the carboxyl terminus of EAA<em>C</em>1 which is required for regulated trafficking. The possibility that this motif targets EAA<em>C</em>1 to an intracellular, "regulated pool" is discussed.

This study investigates the role of platelet-derived microparticles for vascular smooth muscle cell (SMC) proliferation. Microparticles concentration dependently stimulated p42/p44 MAP kinase phosphorylation, c-fos induction, DNA synthesis, and proliferation of cultured bovine coronary artery SMC. The maximum mitogenic effects of microparticles were significantly higher than those of platelet-derived growth factor (PDGF)-BB. Microparticle-induced SMC mitogenesis was heat sensitive, whereas the effects of PDGF were not. In addition, neutralizing anti-PDGF antibodies prevented PDGF-induced DNA synthesis but did not inhibit the effects of microparticles. In contrast to PDGF, which potently stimulated SMC migration, microparticles had only minor migratory activity. These results demonstrate a novel mechanism of SMC mitogenesis by platelet-derived microparticles that is probably independent of PDGF.

The Arf tumor suppressor gene product, p19(Arf), regulates cell proliferation in incipient cancer cells and during embryo development. Beyond its commonly accepted p53-dependent actions, p19(Arf) also acts independently of p53 in both contexts. One such p53-independent effect with in vivo relevance includes its repression of Pdgfrβ, a process that is essential for vision in the mouse. We have utilized cell culture-based and mouse models to define a new role for miR-34a in this process. Ectopic expression of Arf in cultured cells enhanced the expression of several microRNAs predicted to target Pdgfrß synthesis, including the miR-34 family. Because miR-34a has been implicated as a p53-dependent effector, we investigated whether it also contributed to p53-independent effects of p19(Arf). Indeed, in mouse embryo fibroblasts (MEFs) lacking p53, Arf-driven repression of Pdgfrβ and its blockade of Pdgf-B stimulated DNA synthesis were both completely interrupted by anti-microRNA against miR-34a. Ectopic miR-34a directly targeted Pdgfrβ and a plasmid reporter containing wild-type Pdgfrβ 3'UTR sequence, but not one in which the miR-34a target sequence was mutated. Although miR-34a expression has been linked to p53-a well-known effector of p19(Arf)-Arf expression and its knockdown correlated with miR-34a level in MEFs lacking p53. Finally, analysis of the mouse embryonic eye demonstrated that Arf controlled expression of miR-34a, and the related miR-34b and c, in vivo during normal mouse development. Our findings indicate that miR-34a provides an essential link between p19(Arf) and its p53-independent capacity to block cell proliferation driven by Pdgfrβ. This has ramifications for developmental and tumor suppressor roles of Arf.

Platelet-derived growth factor-C (PDGF-C) is a new member of the PDGF family. Its expression in normal and diseased kidney is unknown. Rabbit antisera were generated against human full-length, core domain, and mouse PDGF-C, and their specificity was confirmed by Western blot analyses. Renal PDGF-C expression was analyzed by immunohistochemistry in normal rats (n = 8), mesangioproliferative anti-Thy 1.1 nephritis (n = 4 each at days 1, 4, 6, and 85), passive Heymann nephritis (PHN, n = 4), puromycin nephrosis (PAN, n = 2), Milan normotensive rats (MN, n = 2), and obese Zucker rats (n = 3). PDGF-C expression was also studied in anti-Thy 1.1 rats treated with PDGF-B aptamer antagonists (n = 5) or irrelevant control aptamers (n = 5). PDGF-C was constitutively expressed in arterial smooth muscle cells and collecting duct epithelial cells. Mesangial PDGF-C was markedly upregulated in anti-Thy 1.1 nephritis in parallel with the peak mesangial cell proliferation. Furthermore, PDGF-CC acted as a potent growth factor for mesangial cells in vitro. Inhibition of PDGF-B via specific aptamers reduced the injury in anti-Thy 1.1 nephritis but did not affect the glomerular PDGF-C overexpression or the mitogenicity of PDGF-CC in vitro. In PHN, PAN, and obese Zucker rats, glomeruli remained negative for PDGF-C despite severe glomerular injury. PDGF-C localized to podocytes at sites of focal and segmental sclerosis in MN. Interstitial PDGF-C expression was increased at sites of fibrosing injury in obese Zucker rats. The use of the different antisera resulted in virtually identical findings. It is concluded that PDGF-C is a novel mesangial cell mitogen that is constitutively expressed in the kidney and specifically upregulated in mesangial, visceral epithelial, and interstitial cells after predominant injury to these cells. PDGF-C may therefore be involved in the pathogenesis of renal scarring.

Sphingosine-1-phosphate, a sphingolipid metabolite, is involved in the mitogenic response of platelet-derived growth factor (PDGF) and is formed by activation of sphingosine kinase. We examined the effect of PDGF on sphingosine kinase activation in TRMP cells expressing wild-type or various mutant betaPDGF receptors. Sphingosine kinase was stimulated by PDGF in cells expressing wild-type receptors but not in cells expressing kinase-inactive receptors (R634). Cells expressing mutated PDGF receptors with phenylalanine substitutions at five major tyrosine phosphorylation sites 740/751/771/1009/1021 (F5 mutants), which are unable to associate with PLCgamma, phosphatidylinositol 3-kinase, Ras GTPase-activating protein, or protein tyrosine phosphatase SHP-2, not only failed to increase DNA synthesis in response to PDGF but also did not activate sphingosine kinase. Moreover, mutation of tyrosine-1021 of the PDGF receptor to phenylalanine, which impairs its association with PLCgamma, abrogated PDGF-induced activation of sphingosine kinase. In contrast, PDGF was still able to stimulate sphingosine kinase in cells expressing the PDGF receptor mutated at tyrosines 740/751 and 1009, responsible for binding of phosphatidylinositol 3-kinase and SHP-2, respectively. In agreement, PDGF did not stimulate sphingosine kinase activity in F5 receptor 'add-back' mutants in which association with the Ras GTPase-activating protein, phosphatidylinositol 3-kinase, or SHP-2 was individually restored. However, a mutant PDGF receptor that was able to bind PLCgamma (tyrosine-1021), but not other signaling proteins, restored sphingosine kinase sensitivity to PDGF. These data indicate that the tyrosine residue responsible for binding of PLCgamma is required for PDGF-induced activation of sphingosine kinase. Moreover, calcium mobilization downstream of PLCgamma, but not protein kinase C activation, appears to be required for stimulation of sphingosine kinase by PDGF.-Olivera, A., Edsall, J., Poulton, S., Kazlauskas, A., Spiegel, S. Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.

Renal cell carcinoma (RCC) is a complex disease characterized by mutations in several genes. Loss of function of the von Hippel-Lindau (VHL) tumour suppressor gene is a very common finding in RCC and leads to up-regulation of hypoxia-inducible factor (HIF)-responsive genes accountable for angiogenesis and cell growth, such as platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). Binding of these proteins to their cognate tyrosine kinase receptors on endothelial cells promotes angiogenesis. Promotion of angiogenesis is in part due to the activation of the phosphatidylinositol-3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) pathway. Inhibition of this pathway decreases protein translation and inhibits both angiogenesis and tumour cell proliferation. Although tyrosine kinase inhibitors (TKIs) stand as the main first-line treatment option for advanced RCC, eventually all patients will become resistant to TKIs. Resistance can be overcome by using second-line treatments with different mechanisms of action, such as inhibitors of mTOR, c-MET, programmed death 1 (PD-1) receptor, or the combination of an mTOR inhibitor (mTORi) with a TKI. In this article, we briefly review current evidence regarding mechanisms of resistance in RCC and treatment strategies to overcome resistance with a special focus on the PI3K/AKT/mTOR pathway.

Activation of receptor tyrosine kinases leads to the formation of two different types of plasma membrane structures: peripheral ruffles and dorsal ruffles. Although the formation of both ruffle types requires activation of the small GTPase Rac, the difference in kinetics suggests that a distinct regulatory mechanism operates for their ruffle formation. DOCK1 and DOCK5 are atypical Rac activators and are both expressed in mouse embryonic fibroblasts (MEFs). We found that although PDGF-induced Rac activation and peripheral ruffle formation were coordinately regulated by DOCK1 and DOCK5 in MEFs, DOCK1 deficiency alone impaired dorsal ruffle formation in MEFs. Unlike DOCK5, DOCK1 bound to phosphatidic acid (PA) through the C-terminal polybasic amino acid cluster and was localized to dorsal ruffles. When this interaction was blocked, PDGF-induced dorsal ruffle formation was severely impaired. In addition, we show that phospholipase D, an enzyme that catalyzes PA synthesis, is required for PDGF-induced dorsal, but not peripheral, ruffle formation. These results indicate that the phospholipase D-PA axis selectively controls dorsal ruffle formation by regulating DOCK1 localization.

A series of 3-indoleacrylonitrile tyrphostins, 2-chloro-3-phenylquinolines, and 3-arylquinoxalines were prepared and tested for inhibition of platelet-derived growth factor receptor tyrosine kinase (PDGF-RTK) activity. The potency of the inhibitors was found to be quinoxalines>> quinolines>> indoles. Lipophilic groups (methyl, methoxy) in the 6 and 7 positions and phenyl at the 3 position of quinoxalines and quinolines were essential for potency, in contrast to the hydrophilic catechol group in tyrphostins active against EGFR kinase inhibition at different sites. The inhibitors showed selectivity for PDGF and were not active against EGF receptor and HER-2/c-ErbB-2 receptor.

Oncogenes are important regulators of cancer growth and progression and their action may be modulated by proteins of the growth factor family, such as angiogenic cytokines, known to be strongly involved in neoplastic evolution. Reciprocal interactions between oncogenes and angiogenic modulators may represent, in haematological neoplasms, including multiple myeloma (MM), a possible mechanism of drug resistance. The aim of this work is to investigate in vitro and in vivo whether or not c-myc deregulation is involved in the melphalan resistance elicited by myeloma patients and consequently to clarify the role of the angiogenic factor PDGF-BB in modulating c-myc protein expression. Fifty-one MM patients on chemotherapy with melphalan were analyzed for structural alterations of the c-myc gene, c-Myc protein expression, as well as for serum PDGF-BB release. For the in vitro study, two M14-derived established cell clones, differing for the c-Myc protein expression (c-Myc low -expressing or constitutively expressing clones) were used. Our results show that PDGF-BB is able to up-regulate Myc expression and reduce melphalan sensitivity of tumor cell clones, constitutively expressing c-myc gene product. In addition, down-regulation of c-Myc protein induces the expression of PDGF-beta receptor molecules and reduces PDGF-BB release. In agreement with these results, in vivo data show that melphalan-resistant MM patients present overexpressed c-Myc protein and higher serum PDGF-BB receptor levels compared to minor responding patients.

Although basic fibroblast growth factor (FGF-2) had been shown to inhibit type I collagen gene expression in osteoblast, its inhibitory mechanism is unknown. In the present study, we investigated the underlying mechanisms by which growth factors downregulate type I collagen gene expression. Treatment of mouse osteoblastic MC3T3-E1 cells with okadaic acid (40 ng/ml), an inhibitor of phosphoserine/threonine-specific protein phosphatase and activator of ERK1/2, for 24 h and 48 h completely inhibited steady-state mRNA levels of type I collagen. FGF-2 (30 ng/ml), platelet-derived growth factor-BB (PDGF-BB), 30 ng/ml, and serum, which activate ERK mitogen-activated protein kinase (MAPK) pathway also inhibited collagen type I gene expression, suggesting that the activation of ERK pathway mediates inhibition of type I collagen mRNA. This observation was further confirmed by experiments using inhibitors of the ERK pathway (i.e., PD and U0126), which increased type I collagen mRNA in MC3T3-E1 cells, indicating that the inhibition of ERK pathway upregulates type I collagen gene expression. Low serum (0.3%) markedly increased type I collagen mRNA. MEK inhibitor PD inhibited c-fos induction by FGF-2 and PDGF-BB, suggesting that c-fos is the downstream target of ERK pathway. Our data have clearly demonstrated for the first time that the ERK MAPK pathway play an important role in the regulation of type I collagen gene expression in osteoblastic cells. Results also showed that one of the mechanisms by which FGF-2 and PDGF-BB downregulate type I collagen gene expression in the osteoblast is through the activation of ERK signaling pathway.

The microbial alkaloid staurosporine is a potent but nonselective inhibitor of protein kinases. The derivative CGP 41251 has been shown to exert a high degree of selectivity for inhibition of protein kinase C activity. Both compounds are powerful inhibitors of proliferation of both normal and transformed cells in vitro and exert antitumor efficacy in vivo. In this work we have studied the mode of action of these compounds by analyzing their effects on early events in the induction of proliferation by different growth stimuli. Both drugs blocked the phorbol ester-induced expression of the c-fos proto-oncogene. The effect of CGP 41251 was reversible, since its removal led to a normal expression of c-fos mRNA in response to phorbol 12-myristate 13-acetate. Submicromolar concentrations of CGP 41251 and staurosporine directly inhibited both the platelet-derived growth factor (PDGF) receptor autophosphorylation and the c-fos mRNA expression induced by PDGF stimulation of intact BALB/c 3T3 cells. In contrast, ligand-induced epidermal growth factor receptor autokinase activity in A431 carcinoma cells and epidermal growth factor-dependent c-fos mRNA expression were relatively insensitive to inhibition by CGP 41251. Staurosporine suppressed signal generation by the epidermal growth factor receptor by reducing overall levels of the receptor. We conclude that CGP 41251 is a potent reversible inhibitor of protein kinase C and PDGF-mediated signal transduction. It inhibits the kinase activity of both protein kinase C and the PDGF receptor tyrosine kinase and the subsequent signaling cascade. The broad inhibition of kinases by staurosporine is also reflected at the cellular level and might contribute to the high toxicity of this compound, in comparison to CGP 41251.

Angiogenesis of capillary endothelial cells includes at least four sequential cellular responses: digestion of basement membrane, migration, proliferation, and differentiation. To study differentiation of endothelial cells, we established a brain capillary endothelial cell line from H-2Kb-tsA58 transgenic mice. These cells are stable at 33 degrees C and display endothelial cell-specific characters, such as expression of von Willebrand factor and binding sites for the lectin Bandeiraea simplifolia, and uptake of acetylated-low density lipoprotein. We measured the effects of a panel of growth factors on cellular responses. A number of factors, such as hepatocyte growth factor, vascular endothelial growth factor, and platelet-derived growth factor (PDGF)-AA failed to induce biological responses. PDGF-BB, epidermal growth factor, and acidic and basic fibroblast growth factor (FGF) induced proliferation of the cells. Of all the factors tested, only acidic FGF and basic FGF induced differentiation of the cells, visualized as the formation of tube-like structures of cells grown in three-dimensional collagen gels. All factors were also analyzed for their effects on plasminogen activator (PA)-induction and migration of the cells. Transfected cells, expressing a chimeric receptor, composed of the extracellular part from the PDGF alpha-receptor and the intracellular part from FGF receptor-1, responded to PDGF-AA treatment with plasminogen activator induction, migration, proliferation, and tube formation in collagen. These results indicate that FGF receptor-1 coupled to signal transduction pathways, leading to differentiation. This novel cell model offers the potential of detailed dissection of signal transduction pathways involved in the differentiation of endothelial cells.

Platelet-derived growth factor (PDGF) is a potent mitogen and vasoactive polypeptide for aortic smooth muscle. Because contractile glomerular mesangial cells synthesize a PDGF-like molecule and may respond to PDGF released by infiltrating cells at the site of glomerular inflammation, we studied the effects of exogenous, highly purified PDGF on 1) contraction of cultured rat mesangial cells and 2) membrane phosphoinositide turnover and cytosolic free calcium ([Ca2+]i), as putative mechanisms of membrane signal transduction. PDGF, 10(-11) and 10(-10) M, contracted 56.1 +/- 5.2 and 72.9 +/- 6.4% of the cells, respectively, with an average decrease of cross-sectional area of 22.0 +/- 2.6 and 28.1 +/- 2.7% of basal, as assessed by image-analysis microscopy. PDGF also rapidly increased total water-soluble inositol phosphates, measured after anion-exchange chromatography on perchloric acid-extracted cells, and simultaneously raised [Ca2+]i, measured by the fluorescent intracellular probe fura-2, from basal levels of 83.1 +/- 6.8 to a peak of 229.4 +/- 20.0 nM. We conclude that PDGF stimulates contraction of rat mesangial cells via a phospholipase C-dependent pathway, with potential relevance to the control of glomerular hemodynamics and mesangial proliferation in immune-mediated glomerular disease.

Platelet-derived growth factor (PDGF) has been implicated in the pathobiology of vascular remodeling. The multikinase inhibitor imatinib that targets PDGF receptor (PDGFR), c-kit and Abl kinases, shows therapeutic efficacy against experimental pulmonary hypertension (PH); however, the role of PDGFR-b in experimental PH has not been examined by genetic approach. We investigated the chronic hypoxia-induced PH in mice carrying an activating point mutation of PDGFR-β (D849N) and evaluated the therapeutic efficacy of imatinib. In addition, we studied pulmonary global gene expression and confirmed the expression of identified genes by immunohistochemistry. Chronically hypoxic D849N mice developed PH and strong pulmonary vascular remodeling that was improved by imatinib (100 mg/kg/day) as evident from the significantly reduced right ventricular systolic pressure, right ventricular hypertrophy and muscularization of peripheral pulmonary arteries. Global gene expression analysis revealed that stromal cell derived factor SDF)-1α was significantly upregulated, which was confirmed by immunohistochemistry. Moreover, an enhanced immunoreactivity for SDF-1α, PDGFR-β and CXCR4, the receptor for SDF-1α was localized to the α-smooth muscle cell (SMC) actin positive pulmonary vascular cells in hypoxic mice and patients with idiopathic pulmonary arterial hypertension (IPAH). In conclusion, our findings substantiate the major role of PDGFR activation in pulmonary vascular remodeling by a genetic approach. Immunohistochemistry findings suggest a role for SDF-1α/CXCR4 axis in pulmonary vascular remodeling and point to a potential interaction between the chemokine SDF-1 and the growth factor PDGF signaling. Future studies designed to elucidate an interaction between the chemokine SDF-1 and the PDGF system may uncover novel therapeutic targets.

Prolidase [E.C.3.4.13.9] is a cytosolic exopeptidase that catalyses the hydrolysis of C-terminal proline containing dipeptides or tripeptides. The enzyme plays an important role in the recycling of proline for collagen synthesis. Increase in enzyme activity is correlated with increased rates of collagen turnover but the mechanism and endpoints by which this enzyme is regulated remain largely unknown. We have found that insulin-like growth factor-I (IGF-I), potent stimulator of collagen biosynthesis, induces prolidase activity in cultured human skin fibroblasts. Supporting evidence comes from the following observations: (1) Serum of fasted rats, (IGF-I, 72 +/- 16 ng/ml) showed about 50% reduced ability to stimulate prolidase activity and collagen biosynthesis in confluent fibroblasts in comparison to the effect of control rat serum (IGF-I, 168 +/- 29). (2) An addition of IGF-I (100 ng/ml) to fasted rat serum restored its ability to stimulate prolidase activity and collagen biosynthesis to control values. (3) In confluent human skin fibroblasts, cultured for 48 h with serum free medium prolidase activity was decreased to 50% of control cells, cultured in the presence of normal rat serum. Supplementation of serum free medium with EGF, PDGF and IGF-I (factors that can replace growth promoting activity of serum) stimulated prolidase activity to control values while the medium deprived IGF-I had no such effect. (4) The relative differences in prolidase activity due to specific treatment of confluent cells with above growth factors were accompanied by parallel differences in the amount of the enzyme protein recovered from these cells as shown by western immunoblot analysis. Thus we conclude that prolidase activity is regulated by IGF-I in confluent fibroblasts.

In the present study, we investigated whether proangiogenic growth factors and endothelial progenitor cells (EPCs) induce favourable effects on cutaneous incisional wound healing in diabetic mice. The proangiogenic effects of human EPCs were initially analyzed using a HUVEC in vitro angiogenesis assay and an in vivo Matrigel assay in nude mice (n=12). For the diabetic wound model, 48 Balb/c mice with streptozotocin (STZ)-induced diabetes were divided randomly into 4 groups (12 mice in each group). Subsequently, 3, 5 and 7 days before a 15-mm full-thickness incisional skin wound was set, group 1 was pre-treated subcutaneously with a mixture of vascular endothelial growth factor (VEGF)/basic fibroblast growth factor (bFGF)/platelet-derived growth factor (PDGF) (3.5 µg of each), group 2 with 3.5 µg PDGF and group 3 with an aliquot of two million EPCs, whereas the control animals (group 4) were pre-treated with 0.2 ml saline solution. The wounds were assessed daily and the repaired tissues were harvested 7 days after complete wound closure. The angiogenesis assay demonstrated significantly increased sprout densities, areas and lengths in the EPC-treated group (all p<0.01). In the Matrigel assay, significantly increased microvessel densities, areas and sizes (all p<0.001) were also detected in the EPC-treated group. In the STZ-induced model of diabetes, the animals pre-treated with a combination of proangiogenic factors and EPCs showed in general, a more rapid wound closure. Vessel densities were >2-fold higher in the mice treated with a combination of proangiogenic factors and EPCs (p<0.05) and tensile strengths were higher in the groups treated with proangiogenic growth factors compared to the controls (p<0.05). These results suggest a beneficial effect of pre-treatment with proangiogenic growth factors and EPCs in incisional wound healing.

OBJECTIVE

Krüppel-like factor 4 (KLF4) is implicated in all-trans retinoic acid (ATRA)-induced and platelet-derived growth factor-BB (PDGF-BB)-repressed SM22α expression in vascular smooth muscle cells (VSMCs). However, its exact mechanism of action remains unclear. We determined how KLF4 plays different roles in ATRA- and PDGF-BB-dependent regulation of the SM22α gene.

RESULTS

ATRA and PDGF-BB induced KLF4 expression but exhibited an opposite effect on SM22α expression and VSMC proliferation. Chromatin immunoprecipitation and oligonucleotide pull-down assays showed that KLF4 was directly bound to the KLF4 binding sites 1 ((-263)CACCC(-259)) and 2 ((-136)GTGGG(-132)) of the SM22α promoter. ATRA increased the binding of KLF4 to site 2, whereas PDGF-BB decreased the binding of KLF4 to site 1. ATRA stimulated KLF4 acetylation by inducing KLF4 phosphorylation and increasing its interaction with p300 via activating c-Jun NH(2)-terminal kinase (JNK) and p38 pathways, and acetylated KLF4 increased its binding activity to site 2. PDGF-BB stimulated KLF4 deacetylation by inducing KLF4 dephosphorylation and increasing its interaction with histone deacetylase 2 (HDAC2) via activating extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathways, and deacetylated KLF4 dissociated from site 1.

CONCLUSIONS

In VSMCs, ATRA activates and PDGF-BB represses SM22α expression through KLF4 binding to, or dissociating from, its different cis-elements in an acetylation-dependent manner.